Culturing Human Gut Microbiomes in the Laboratory.

The human gut microbiota is a complex community of prokaryotic and eukaryotic microbes and viral particles that is increasingly associated with many aspects of host physiology and health. However, the classical microbiology approach of axenic culture cannot provide a complete picture of the complex interactions between microbes and their hosts in vivo. As such, recently there has been much interest in the culture of gut microbial ecosystems in the laboratory as a strategy to better understand their compositions and functions. In this review, we discuss the model platforms and methods available in the contemporary microbiology laboratory to study human gut microbiomes, as well as current knowledge surrounding the isolation of human gut microbes for the potential construction of defined communities for use in model systems.

Impact of food preservatives based on immobilized phenolic compounds on an in vitro model of human gut microbiota.

To address concerns about the biocompatibility of novel phenolic immobilization-based food preservatives, their impact on the composition and metabonomic profile of a defined community of human gut microbiota was evaluated. Three phenolics (eugenol, vanillin and ferulic acid) presented in two forms (free or immobilized on different supports) were tested at two concentration levels (0.5 and 2 mg/mL). Free eugenol was the phenolic with the greatest impact on gut microbiota, with a remarkable increase in the abundance of Lachnospiraceae and Akkermansiaceae families. In contrast, immobilized phenolics produced an increase in the abundance of Bacteroides with a reduction in the ratio of Firmicutes to Bacteroidetes. The metabonomic profile was also affected by free and immobilized phenolics differently in terms of fermentation by-products and phenolic biotransformation metabolites. Thus the results suggest the importance of evaluating the impact of new compounds or materials added to food on human gut microbiota and their potential use to modulate microbiota composition.

The Robogut: A Bioreactor Model of the Human Colon for Evaluation of Gut Microbial Community Ecology and Function.

The human colon is inhabited by a complex community of microbes. These microbes are integral to host health and physiology. Understanding how and when the microbiome causally influences host health will require microbiome models that can be tightly controlled and manipulated. While in vivo models are unrivalled in their ability to study host-microbial interplay, in vitro models are gaining in popularity as methods to study the ecology and function of the gut microbiota, and benefit from tight controllability and reproducibility, as well as reduced ethical constraints. In this set of protocols, we describe the Robogut, a single-stage bioreactor system designed to replicate the conditions of the distal human colon, to culture whole microbial communities derived from stool and/or colonic biopsy samples, with consideration of methods to create culture medium formulations and to build, run, and sample the bioreactor apparatus. Cleaning and maintenance of the bioreactor system are also described. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Growth medium preparation Support Protocol 1: Preparing medium supplements Basic Protocol 2: Preparing the bioreactor vessels Support Protocol 2: Making acid and base bottles Support Protocol 3: Preparing the effluent bottles Support Protocol 4: Making acid solution Support Protocol 5: Making base solution Basic Protocol 3: Preparing inoculum and inoculating bioreactors Alternate Protocol 1: Preparing inoculum less than 0.5% (w/v) of vessel volume Alternate Protocol 2: Preparing synthetic community aliquots and inoculation via the septum Alternate Protocol 3: Preparing inoculum from a tissue sample Basic Protocol 4: Sampling the bioreactor vessel Basic Protocol 5: Harvesting bioreactor vessel contents at end of experiment Support Protocol 6: Cleaning and sterilizing sampling needles Basic Protocol 6: Cleaning the bioreactor vessel Support Protocol 7: Cleaning bioreactor support bottles.

Dietary Nitrate and Corresponding Gut Microbiota Prevent Cardiac Dysfunction in Obese Mice.

Impaired heart function can develop in individuals with diabetes in the absence of coronary artery disease or hypertension, suggesting mechanisms beyond hypertension/increased afterload contribute to diabetic cardiomyopathy. Identifying therapeutic approaches that improve glycemia and prevent cardiovascular disease are clearly required for clinical management of diabetes-related comorbidities. Since intestinal bacteria are important for metabolism of nitrate, we examined whether dietary nitrate and fecal microbial transplantation (FMT) from nitrate-fed mice could prevent high-fat diet (HFD)-induced cardiac abnormalities. Male C57Bl/6N mice were fed a low-fat diet (LFD), HFD, or HFD+Nitrate (4 mmol/L sodium nitrate) for 8 weeks. HFD-fed mice presented with pathological left ventricle (LV) hypertrophy, reduced stroke volume, and increased end-diastolic pressure, in association with increased myocardial fibrosis, glucose intolerance, adipose inflammation, serum lipids, LV mitochondrial reactive oxygen species (ROS), and gut dysbiosis. In contrast, dietary nitrate attenuated these detriments. In HFD-fed mice, FMT from HFD+Nitrate donors did not influence serum nitrate, blood pressure, adipose inflammation, or myocardial fibrosis. However, microbiota from HFD+Nitrate mice decreased serum lipids, LV ROS, and similar to FMT from LFD donors, prevented glucose intolerance and cardiac morphology changes. Therefore, the cardioprotective effects of nitrate are not dependent on reducing blood pressure, but rather mitigating gut dysbiosis, highlighting a nitrate-gut-heart axis. ARTICLE HIGHLIGHTS: Identifying therapeutic approaches that prevent cardiometabolic diseases are clearly important, and nitrate represents one such potential compound given its multifactorial metabolic effects. We aimed to determine whether nitrate could prevent high-fat diet (HFD)-induced cardiac abnormalities and whether this was dependent on the gut microbiome. Dietary nitrate attenuated HFD-induced pathological changes in cardiac remodelling, left ventricle reactive oxygen species, adipose inflammation, lipid homeostasis, glucose intolerance, and gut dysbiosis. Fecal microbial transplantation from nitrate-fed mice also prevented serum dyslipidemia, left ventricle reactive oxygen species, glucose intolerance, and cardiac dysfunction. Therefore, the cardioprotective effects of nitrate are related to mitigating gut dysbiosis, highlighting a nitrate-gut-heart axis.

Influence of free and immobilized chitosan on a defined human gut microbial ecosystem.

In this work, the influence of different forms of presentation of chitosan in the human gut microbiota with a defined bacterial community was evaluated. First, the susceptibility of individual gut bacterial isolates against chitosan was studied within a concentration range between 0.125 and 1 mg/mL. Then, the impact of chitosan (0.25 and 1 mg/mL) on a defined human gut microbial ecosystem was studied by metagenomic and metabonomic analyses. The results showed that chitosan in its free form had a high impact on individual isolates with a minimum inhibitory concentration below 1 mg/mL for most of the strains studied. In comparison, chitosan immobilized in the different carriers displayed a diverse effect on gut microbiota. The most susceptible strains were Agathobacter rectalis strain 16-6-I 1 FAA, Clostridium spiroforme strain 16-6-I 21 FAA and Mediterraneibacter faecis strain 16-6-I 30 FAA. The impact of the different modes of presentation of chitosan was strain-specific and species-specific when compared to results obtained from analysis of other strains within the genera Agathobacter, Clostridium and Mediterraneibacter, and therefore a study using a defined ecosystem was needed to extrapolate the results. Significant decreases in defined community richness and diversity and changes in metabolic profile were observed after exposure to free chitosan. Free chitosan produced significant reductions in the abundance of the genera Lachnoclostridium, Anaerotignum, Blautia, Enterococcus, Eubacterium and Ruthenibacterium together with a slight decrease of the production of SCFAs, among other fermentation by-products. The immobilized chitosan significantly alleviated the impact caused by the antimicrobial polymer and significantly increased the relative abundance of the Bacteroidetes phylum compared to free chitosan. These results suggest the significance of assessing the impact of new ingredients and materials included in food on the human gut microbiota with models that simulate the gastrointestinal environment, such as in vitro bioreactor systems.

Metagenomics-Based, Strain-Level Analysis of Escherichia coli From a Time-Series of Microbiome Samples From a Crohn's Disease Patient.

Dysbiosis of the gut microbiome, including elevated abundance of putative leading bacterial triggers such as E. coli in inflammatory bowel disease (IBD) patients, is of great interest. To date, most E. coli studies in IBD patients are focused on clinical isolates, overlooking their relative abundances and turnover over time. Metagenomics-based studies, on the other hand, are less focused on strain-level investigations. Here, using recently developed bioinformatic tools, we analyzed the abundance and properties of specific E. coli strains in a Crohns disease (CD) patient longitudinally, while also considering the composition of the entire community over time. In this report, we conducted a pilot study on metagenomic-based, strain-level analysis of a time-series of E. coli strains in a left-sided CD patient, who exhibited sustained levels of E. coli greater than 100X healthy controls. We: (1) mapped out the composition of the gut microbiome over time, particularly the presence of E. coli strains, and found that the abundance and dominance of specific E. coli strains in the community varied over time; (2) performed strain-level de novo assemblies of seven dominant E. coli strains, and illustrated disparity between these strains in both phylogenetic origin and genomic content; (3) observed that strain ST1 (recovered during peak inflammation) is highly similar to known pathogenic AIEC strains NC101 and LF82 in both virulence factors and metabolic functions, while other strains (ST2-ST7) that were collected during more stable states displayed diverse characteristics; (4) isolated, sequenced, experimentally characterized ST1, and confirmed the accuracy of the de novo assembly; and (5) assessed growth capability of ST1 with a newly reconstructed genome-scale metabolic model of the strain, and showed its potential to use substrates found abundantly in the human gut to outcompete other microbes. In conclusion, inflammation status (assessed by the blood C-reactive protein and stool calprotectin) is likely correlated with the abundance of a subgroup of E. coli strains with specific traits. Therefore, strain-level time-series analysis of dominant E. coli strains in a CD patient is highly informative, and motivates a study of a larger cohort of IBD patients.